short report
Haematologica 1994; 79:452-455
CD11c EXPRESSION IN B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA.
A COMPARISON OF RESULTS OBTAINED WITH DIFFERENT
MONOCLONAL ANTIBODIES
Stefano Molica, Angela Dattilo, Ada Mannella, Domenico Levato
Divisione di Ematologia, Ospedale Regionale A. Pugliese, Catanzaro, Italy
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ABSTRACT
In order to obtain more information on the pattern of CD11c-positivity in otherwise typical Bcell chronic lymphocytic leukemia (CLL), we analyzed immunological and clinico-pathological
features of 99 such patients studied with two different monoclonal antibodies (MoAbs). Fifty-two
out of 70 (74.2%) patients stained with IOM-11C MoAb and 3 out of 29 (10.3%) patients stained
with Leu-M5 MoAb had more than 30% CD11c positive cells (P < 0.0002). The two groups were
similar with regard to the expression of B-cell CLL-related antigens (CD5, CD20, CD23), as well as
clinico-pathological features (i.e. Binet clinical stage and pattern of bone marrow involvement),
thus suggesting that differences in CD11c expression were due to different reactivity patterns of
the MoAbs utilized. In our experience, the use of different reagents may affect immunophenotyping results, thus providing conflicting data at times.
Key words: CD11c, B-CLL, expression pattern
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D11c, the 150kd molecular weight achain of a two-chain (150 and 95 kd)
glycoprotein, belongs to the integrin
family of glycoproteins that are members of the
families of cellular adhesion antigens.1 In its
original description, CD11c showed strong
reactivity with hairy cells 2 and, when coexpressed with CD22, was proposed as a unique
marker of hairy cell leukemia (HCL).3
More recently, several studies have shown that
CD11c is expressed in 13% to 78% of otherwise
typical CLL patients; these results reflect differences in patient selection, in the monoclonal
antibodies (MoAbs) used to detect CD11c, and
in the technical sensitivity of flow cytometry
instrumentations.4,9 Keeping these concepts in
mind, we analyzed CD11c-expression in 99
CD5+ B-cell CLL patients diagnosed in two different periods. For the purpose of the present
study the patient population was split into two
groups according to the commercial MoAb used
to detect CD11c (Leu-M5 or IOM 11c). Since
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the clinico-hematological features of the two
groups were alike, differences in CD11c expression were considered suggestive of a different
pattern of positivity of the MoAbs utilized.
Materials and Methods
Patient characteristics
Ninety-nine patients diagnosed as having
CLL at our institution during the period June
1990 to March 1994 form the basis of this
study. The mean age was 66 years (SD, 8.4) and
the male to female ratio 61 to 38.
B-CLL was diagnosed according to generally
accepted criteria that included peripheral
blood lymphocytosis greater than 5u109/L and
bone marrow (BM) lymphocytosis greater than
30%. Diagnosis of B-cell CLL was confirmed in
all instances by analysis of surface Ig light
chain and CD5 expression. All patients were
staged according to the Binet clinical staging
Correspondence: Dr. Stefano Molica, via Casalinuovo 6, 88100 Catanzaro, Italy. Tel. international +39.961.883001. Fax. international
+39.961.740055.
Received March 14, 1994; accepted July 15, 1994.
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Immunophenotype analyses
Fresh peripheral blood samples were used for
immunological analyses. Mononuclear cells
were separated from whole blood by FycollHypaque density gradient centrifugation, and
cells were stained using both direct and indirect
techniques. In the latter cases, a second-layer
reagent of fluorescence (FTIC)-conjugated F
(ab’)2 fragment of goat anti-mouse immunoglobulins (DAKO, Denmark) was used.
Monoclonal antibodies employed along with
their CD as defined by the 4th International
Workshop on Leukocyte and Differentiation
Antigens, were as follows: HLA-DR (anti MHC-
class II), OKT3-CD3 (T-lymphocytes), OKCLLCD5 (T-lymphocytes, B-lymphocyte subsets, BCLL cells), OKT26a-CD25 (activated T and B
cells, activated macrophages), OKBCALLACD10 (lymphoid progenitors, C-ALL, granulocytes), Leu12-CD19 (immature and mature Blymphocytes, malignant B-cells), Leu16-CD20
(B-lymphocytes, malignant B-cells), Leu20CD23 (activated B-cells, B-CLL), κ and λ light
chains (SIg) (Ortho, Raritan, NJ).
As far as CD11c is concerned, two different
commercial MoAbs were used. In 70 patients
diagnosed during the period June 1990 to March
1993, we utilized an IOM-11c MoAb (Immunotech, Marseille, France); in the remaining
29 patients, diagnosed between August 1993 and
March 1994, Leu-M5 MoAb (Bekton-Dickinson,
Mountain View, CA) was utilized. Flow cytometric measurements using simple color immunofluorescence were made by means of a
CYTORON cytofluorograph (Ortho Diagnostic
System).
Because of the different right and forward
angle light-scattering properties of blood cells,
only lymphocytes were used to separate this cell
fraction from others. Controls were provided by
cells incubated directly with FTIC-labeled second reagent alone, or by cells incubated with an
unrelated MoAb with an isotype identical to that
of the test MoAb (IgG1, CD41; IgG2a, CD10;
IgG2b, CD69).
Leukemic cells were counted via the expression of CD19 and residual T-cells were identified through the expression of surface CD3
molecules. According to this criteria, the mean
value of CD19+ was 81.7±13.3% and that of
residual T-cells 14.6±11.5%.
Thus, the requirement for CD11c positivity
was expressed by at least 30% of positive
leukemic cells. In 22 out of 99 patients who had
a residual T-cell population greater than 20%,
CD11c-positivity was assessed with double fluorescence. For this analysis phycoerythrinated
(CD19/PE) and fluorescinated MoAbs (CD11cFTIC) were used. Mean fluorescence intensity
(MFI) was assessed on the basis of the mean
channel fluorescence of each positive sample
(linear acquisition, 0 to 250 channels).
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system10 (stage A, 58; stage B, 23; stage C, 18),
and the pattern of BM involvement was evaluated by means of BM biopsy in 62 of the 99
patients. Biopsy was not performed in the
remaining 37 patients mainly due to lack of
informed consent. Four histological patterns
were recognized: interstitial, 32; nodular, 11;
mixed, 4; diffuse, 15.11,12 For the purpose of the
present study, interstitial, nodular and mixed
were grouped together as the non-diffuse pattern (Table 1).
Clinical stage
A
B
C
BM histology
Non diffuse
Diffuse
Group A
Group B
70
29
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Table 1. CD11c expression in B-CLL. Immunological and
clinico-pathological characteristics of patients studied with
IOM-11C MoAb (Group A) and Leu-M5 MoAb (Group B).
P
40 (57.1 %)
17 (24.2%)
13 (18.5%)
18 (62%)
6 (20.6%)
5 (17.2%)
NS
36 (75%)
12 (25%)
11 (78.5%)
3 (21.4%)
NS
74.2%
100%
87.2%
92.2%
65.9%
23.4%
10.6%
10.3%
100%
89%
95.6%
72.2%
13.6%
13.6%
<0.0002
NS
NS
NS
NS
NS
NS
Cases
CD11c+
CD5+
CD20+
CD23+
κ+
λ+
SIg BDL*
*SIg BDL: SIg below detection level
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CD11 in B-cell
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groups were alike with respect to the expression
of CLL B-lineage antigens such as CD19, CD20,
CD23, and light chains. This was also true when
clinico-pathological comparisons were made
according to clinical stage and histological pattern of BM involvement.
CD11c always displayed low fluorescence
intensity (MFI, 86.5±15.3). This pattern of
CD11c positivity can be distinguished from
that of HCL cells. Indeed, this differentiation is
easily made when CD11c histograms from a BCLL and an HCL patient, respectively (Figure
1), are compared.
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Discussion
The expression of CD11c in CLL is debated,
and CD11c antibodies are heterogeneous and
give varying results with CLL cells. In this setting we compared the results of CD11c expression obtained with two different commercial
MoAbs. Since the two patient populations were
alike with regard to clinico-hematological as
well as immunological features, differences in
CD11c expression were considered representative of different reactivity patterns of the MoAbs
utilized. Our results are in keeping with those of
literature; overall 140 out of 378 (37%) B-cell
CLL patients analyzed with Leu-M5 and 108 out
140 (77.1%) analyzed with IOM-11c MoAb
expressed CD11c (p < 0.0002).3,9
Finally, the present report provides information that is lacking in the literature on the positivity pattern of different CD11c MoAbs in Bcell CLL. In our opinion, when immunophenotyping results are compared it is mandatory to
take into account the reagents used.
Figure 1. CD11c expression in B-cell CLL patient (A; MFI,
88.9 + 9.9) and hairy cell leukemia patient (B; MFI, 168.3
+ 28.4).
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Statistical analyses
The Student’s t-test was used to evaluate differences among the means of various phenotypic groups. When dealing with discrete variables, statistical analyses were carried out by
means of the chi-square test.
Results
Fifty-two out of 70 (74.2%) patients stained
with IOM-11c MoAb and 3 out of 29 (10.3%)
stained with Leu-M5 MoAb had more than 30%
CD11-positive cells (p<0.0002). When patients
were stratified according to the MoAb utilized
for CD11c detection, it was clear that the two
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